Multi-biomarker approach for the (eco)toxicity of UV-filter environmental pollution on the Mediterranean mussel Mytilus galloprovincialis in a multiple stressor context. The case of 4-MBC under salinity shifts.

Marine-coastal ecosystems are rapidly transforming because of climate change (CC). At the same time, the impacts of emerging organic contaminants (i.e., organic UV-filters) on these ecosystems are intensifying. In the Mediterranean, the consequences of these disturbances are occurring at a fast pace making this area a potential sentinel site to be investigated. While singular effects of organic UV-filters or CC-related factors on marine biota have been relatively described, their combined impact is still largely unknown. Thus, the objective of this study was to assess the long-term responses of the Mediterranean mussel Mytilus galloprovincialis towards anticipated salinity changes (decreases-S20 or increases-S40) when exposed to environmentally relevant concentrations of the UV-filter 4-methylbenzylidene camphor (4-MBC). An integrated multi-biomarker approach was applied, featuring general and oxidative stress, antioxidant and biotransformation enzyme capacity, energy metabolism, genotoxicity, and neurotoxicity biomarkers. Results showed that both projected salinities, considered separately, exerted non-negligible impacts on mussels' health status, with greater biological impairments found at S 40. Combining both stressors resulted in an evident increase in mussels' susceptibility to the UV-filter, which exacerbated the toxicity of 4-MBC. The dominant influence of salinity in the climate change-contaminant interaction played a crucial role in this outcome. The most severe scenario occurred when S 20 was combined with 4-MBC. In this situation, mussels exhibited a decrease in filtration rate, metabolic capacity and deployment of energy reserves increased, with an upregulation of biotransformation and inhibition of antioxidant enzyme activities. This exposure also led to the observation of cellular and DNA damage, as well as an increase in AChE activity. Furthermore, salinity-dependent bioaccumulation patterns were evaluated revealing that the lowest values in contaminated mussels are found at S 20. Overall, the present findings provide evidence that projected CC/pollutant scenarios may represent high risks for mussels' populations, with global relevant implications for the ecosystem level.

Toxicity of ionic liquids in marine and freshwater microorganisms and invertebrates: state of the art.

The variety of applications and expected growth in ionic liquid production are raising concerns about the release of these compounds into aquatic systems. Up to date, 103 studies have provided ecotoxicological data regarding the exposure effects of Ionic Liquids towards aquatic microorganisms and invertebrate species: 61 were devoted to freshwater species (n = 28), while marine species (n = 12) were mentioned in 42. The aim of this review, by gathering published studies on ionic liquids and model aquatic organisms, was to present the toxic effects described in distinct species and to understand which are the main factors influencing the toxicity of some ionic liquids. In accordance with the most recognized pattern, freshwater species were featured in a higher number of publications than marine ones. After literature analysis, algal species were the most represented organisms in aquatic toxicity assessments. Among tested compounds, the imidazolium cations in combination with long alkyl-chain anions, showed to be the most toxic one. In analytical terms, it is not straightforward to find the undissociated compound in a natural compartment, as ionic liquids are composed of ionic components, easily subjected to dissociation. Given the aforementioned, the present review paper points out the need of increasing the number of organisms being assessed in ionic liquids toxicity assays, in order to start defining monitoring procedures. Moreover, such would allow a better understanding of ionic liquids contamination status and, also, the opportunity to remark the effectiveness of new in silico methods for the ecotoxicity assessment of this kind of substances.

Posidonia oceanica (L.) (Delile, 1813) extracts as a potential booster biocide in fouling-release coatings.

Since the banning of tributyltin, the addition of inorganic (metal oxides) and organic (pesticides, herbicides) biocides in antifouling paint has represented an unavoidable step to counteract biofouling and the resulting biodeterioration of submerged surfaces. Therefore, the development of new methods that balance antifouling efficacy with environmental impact has become a topic of great importance. Among several proposed strategies, natural extracts may represent one of the most suitable alternatives to the widely used toxic biocides. Posidonia oceanica is one of the most representative organisms of the Mediterranean Sea and contains hundreds of bioactive compounds. In this study, we prepared, characterized, and assessed a hydroalcoholic extract of P. oceanica and then compared it to three model species. Together, these four species belong to relevant groups of biofoulers: bacteria (Aliivibrio fischeri), diatoms (Phaeodactylum tricornutum), and serpulid polychaetes (Ficopomatus enigmaticus). We also added the same P. oceanica extract to a PDMS-based coating formula. We tested this coating agent with Navicula salinicola and Ficopomatus enigmaticus to evaluate both its biocidal performance and its antifouling properties. Our results indicate that our P. oceanica extract provides suitable levels of protection against all the tested organisms and significantly reduces adhesion of N. salinicola cells and facilitates their release in low-intensity waterflows.

Polyethylene Glycol-b-poly(trialkylsilyl methacrylate-co-methyl methacrylate) Hydrolyzable Block Copolymers for Eco-Friendly Self-Polishing Marine Coatings.

Hydrolyzable block copolymers consisting of a polyethylene glycol (PEG) first block and a random poly(trialkylsilyl methacrylate (TRSiMA, R = butyl, isopropyl)-co-methyl methacrylate (MMA)) second block were synthesized by RAFT polymerization. Two PEGs with different molar masses (Mn = 750 g/mol (PEG1) and 2200 g/mol (PEG2)) were used as macro-chain transfer agents and the polymerization conditions were set in order to obtain copolymers with a comparable mole content of trialkylsilyl methacrylate (~30 mole%) and two different PEG mole percentages of 10 and 30 mole%. The hydrolysis rates of PEG-b-(TRSiMA-co-MMA) in a THF/basic (pH = 10) water solution were shown to drastically depend on the nature of the trialkylsilyl groups and the mole content of the PEG block. Films of selected copolymers were also found to undergo hydrolysis in artificial seawater (ASW), with tunable erosion kinetics that were modulated by varying the copolymer design. Measurements of the advancing and receding contact angles of water as a function of the immersion time in the ASW confirmed the ability of the copolymer film surfaces to respond to the water environment as a result of two different mechanisms: (i) the hydrolysis of the silylester groups that prevailed in TBSiMA-based copolymers; and (ii) a major surface exposure of hydrophilic PEG chains that was predominant for TPSiMA-based copolymers. AFM analysis revealed that the surface nano-roughness increased upon immersion in ASW. The erosion of copolymer film surfaces resulted in a self-polishing, antifouling behavior against the diatom Navicula salinicola. The amount of settled diatoms depended on the hydrolysis rate of the copolymers.

The influence of salinity on the toxicity of chemical UV-filters to sperms of the free-spawning mussel Mytilus galloprovincialis (Lamark, 1819).

Marine-coastal systems have been increasingly exposed to multiple stressors, including anthropogenic pollution and variations of Climate Change (CC) related factors, whose coexistence could create associated environmental and ecotoxicological risks. Among emergent stressors, 4-methylbenzylidenecamphor (4-MBC) and benzophenone-3 (BP-3) UV-filters are compounds widely used in increasing consumer products, resulting in their ubiquity in aquatic environments and possible pressing challenges on gamete susceptibility. Since most marine invertebrates reproduce by external fertilization, after spawning, gametes may be exposed to several pressures, affecting reproductive success and outcome. The present study focuses on the spermiotoxicity of the environmentally relevant UV-filters 4-MBC and BP-3 combined with salinity shifts, as potential modulators of their harmful effects. For this, Mytilus galloprovincialis male gametes were exposed in vitro to environmentally relevant and slightly higher concentrations (1, 10 and 100 µg/L) of 4-MBC or BP-3 under three different salinities (S 20, 30 and 40). Sperm quality endpoints associated with oxidative status, viability, motility, kinetics, and genotoxicity were evaluated. Similarities and differences in sperm responses among all conditions were highlighted by principal coordinates analysis (PCO). Results showed that salinity acting alone posed greater sperms impairments at the lowest (20) and highest (40) tested levels. When salinity acts as a co-varying stressor, salinity-dominant interactive effects resulted evident, especially for 4-MBC at S 40 and BP-3 at S 20. These findings were pointed out as the worst exposure conditions for M. galloprovincialis sperms, since caused major toxicological effects in terms of: (I) oxidative stress, sperm structural impairments, motility and kinetic alterations in 4-MBC-exposed sperms; (II) DNA damage, compromised mitochondrial activity and hyperactivation in BP-3-exposed ones. Overall, it stands out that salinity influences UV-filter toxicological pathways and, thereby, the potential environmental risk of these contaminants on M. galloprovincialis male gametes, especially in an expected salinity stress scenario.

A multi-bioassay integrated approach to assess antifouling potential of extracts from the Mediterranean sponge Ircinia oros.

The phylum Porifera and their symbionts produce a wide variety of bioactive compounds, playing a central role in their ecology and evolution. In this study, four different extracts (obtained by non-polar and semi-polar extraction methodologies) of the Mediterranean sponge Ircinia oros were tested through a multi-bioassay integrated approach to assess their antifouling potential. Tests were performed using three common species, associated with three different endpoints: the marine bacterium Aliivibrio fischeri (inhibition of bioluminescence), the marine diatom Phaeodactylum tricornutum (inhibition of growth), and different development stages of the brackish water serpulid Ficopomatus enigmaticus (gametes: sperm motion, vitality inhibition and cellular damage; larvae: development; adults: AChE (acetylcholinesterase)-inhibitory activity). The effects of extracts were species specific and did not vary among different extraction methodologies. In particular, no significant reduction of bioluminescence of A. fischeri was observed for all tested samples. By contrast, extracts inhibited P. tricornutum growth and had toxic effects on different F. enigmaticus' developmental stages. Our results suggest that the proposed test battery can be considered a suitable tool as bioactivity screening of marine natural products.

Amphiphilic Polyphosphonate Copolymers as New Additives for PDMS-Based Antifouling Coatings.

Poly(ethyl ethylene phosphonate)-based methacrylic copolymers containing polysiloxane methacrylate (SiMA) co-units are proposed as surface-active additives as alternative solutions to the more investigated polyzwitterionic and polyethylene glycol counterparts for the fabrication of novel PDMS-based coatings for marine antifouling applications. In particular, the same hydrophobic SiMA macromonomer was copolymerized with a methacrylate carrying a poly(ethyl ethylene phosphonate) (PEtEPMA), a phosphorylcholine (MPC), and a poly(ethylene glycol) (PEGMA) side chain to obtain non-water soluble copolymers with similar mole content of the different hydrophilic units. The hydrolysis of poly(ethyl ethylene phosphonate)-based polymers was also studied in conditions similar to those of the marine environment to investigate their potential as erodible films. Copolymers of the three classes were blended into a condensation cure PDMS matrix in two different loadings (10 and 20 wt%) to prepare the top-coat of three-layer films to be subjected to wettability analysis and bioassays with marine model organisms. Water contact angle measurements showed that all of the films underwent surface reconstruction upon prolonged immersion in water, becoming much more hydrophilic. Interestingly, the extent of surface modification appeared to be affected by the type of hydrophilic units, showing a tendency to increase according to the order PEGMA < MPC < PEtEPMA. Biological tests showed that Ficopomatus enigmaticus release was maximized on the most hydrophilic film containing 10 wt% of the PEtEP-based copolymer. Moreover, coatings with a 10 wt% loading of the copolymer performed better than those containing 20 wt% for the removal of both Ficopomatus and Navicula, independent from the copolymer nature.

Anisakid and Raphidascaridid parasites in Trachurus trachurus: infection drivers and possible effects on the host's condition.

This study investigated the distribution of nematode larvae of Anisakidae and Raphidascarididae (genera Anisakis and Hysterothylacium) in Trachurus trachurus (Linnaeus, 1758) in the Ligurian and central-northern Tyrrhenian Seas. The relationship between the number of parasites and the length and weight parameters of the fish was assessed, and the possible effect of the parasites on the condition factor was evaluated. A total of 190 T. trachurus specimens were collected in July 2019. Parasites were found in 70 individuals. A total of 161 visible larvae were collected in the viscera. Morphological analysis revealed the presence of Anisakis spp. in 55 fish and Hysterothylacium spp. in 15 fish, while 5 fish showed coinfection with both genera. The specimens subjected to PCR (n = 67) showed that 85% of the Anisakis larvae analyzed belonged to the species A. pegreffii, while the remaining 15% belonged to hybrids of A. pegreffii-A. simplex (s.s.). A total of 58% (n = 7) of the Hysterothylacium larvae analyzed belonged to the species H. fabri, while 42% belonged to the species H. aduncum. Our results support the hypothesis that infection with these parasites does not affect the condition of the fish host analyzed, and that body size and depth are major drivers in determining infection levels with Anisakid and Raphidascaridid nematodes.

Sperm quality assessment in Ficopomatus enigmaticus (Fauvel, 1923): Effects of selected organic and inorganic chemicals across salinity levels.

Contamination by organic and inorganic compounds remains one of the most complex problems in both brackish and marine environments, causing potential implications for the reproductive success and survival of several broadcast spawners. Ficopomatus enigmaticus is a tubeworm polychaete that has previously been used as a model organism for ecotoxicological analysis, due to its sensitivity and ecological relevance. In the present study, the effects of five trace elements (zinc, copper, cadmium, arsenic and lead), one surfactant (sodium dodecyl sulfate, SDS) and one polycyclic aromatic hydrocarbon (benzo(a)pyrene, B(a)P) on the sperm quality of F. enigmaticus were investigated. Sperm suspensions were exposed in vitro to different concentrations of each selected contaminant under four salinity conditions (10, 20, 30, 35). Possible adverse effects on sperm function were assessed by measuring oxidative stress, membrane integrity, viability and DNA damage. Sperm quality impairments induced by organic contaminants were more evident than those induced by inorganic compounds. SDS exerted the largest effect on sperm. In addition, F. enigmaticus sperm showed high tolerance to salinity variation, supporting the wide use of this species as a promising model organism for ecotoxicological assays. Easy and rapid methods on polychaete spermatozoids were shown to be effective as integrated sperm quality parameters or as an alternative analysis for early assessment of marine and brackish water pollution.

Soluble esterases as biomarkers of neurotoxic compounds in the widespread serpulid Ficopomatus enigmaticus (Fauvel, 1923).

The characterization of soluble cholinesterases (ChEs) together with carboxylesterases (CEs) in Ficopomatus enigmaticus as suitable biomarkers of neurotoxicity was the main aim of this study. ChEs of F. enigmaticus were characterized considering enzymatic activity, substrate affinity (acetyl-, butyryl-, propionylthiocholine), kinetic parameters (Km and Vmax) and in vitro response to model inhibitors (eserine hemisulfate, iso-OMPA, BW284C51), and carbamates (carbofuran, methomyl, aldicarb, and carbaryl). CEs were characterized based on enzymatic activity, kinetic parameters and in vitro response to carbamates (carbofuran, methomyl, aldicarb, and carbaryl). Results showed that cholinesterases from F. enigmaticus showed a substrate preference for acetylthiocholine followed by propionylthiocholine; butyrylthioline was not hydrolyzed differently from other Annelida species. CE activity was in the same range of cholinesterase activity with acetylthiocholine as substrate; the enzyme activity showed high affinity for the substrate p-nytrophenyl butyrate. Carbamates inhibited ChE activity with propionylthiocholine as substrate to a higher extent than with acetylthiocoline. Also CE activity was inhibited by all tested carbamates except carbaryl. In vitro data highlighted the presence of active forms of ChEs and CEs in F. enigmaticus that could potentially be inhibited by pesticides at environmentally relevant concentration.

Ecotoxicological properties of ketoprofen and the S(+)-enantiomer (dexketoprofen): Bioassays in freshwater model species and biomarkers in fish PLHC-1 cell line.

The increased use of nonsteroidal anti-inflammatory drugs (NSAIDs) has resulted in their ubiquitous presence in the environment. The toxicological properties of these 2 widely prescribed NSAIDs, namely racemic ketoprofen and its enantiomer S(+)-ketoprofen (dexketoprofen), were evaluated, firstly, by acute and chronic toxicity tests using 3 representative model organisms (Vibrio fischeri, Pseudokirchneriella subcapitata, and Ceriodaphnia dubia) and, secondly, by evaluating the responses of biotransformation systems and multidrug resistance-associated proteins (MRP1/MRP2) using the Poeciliopsis lucida hepatocellular carcinoma 1 (PLHC-1) fish hepatic cell line. Toxicity data from both acute and chronic dexketoprofen exposure indicated higher sensitivity through inhibition of bioluminescence and algal growth and through increased mortality/immobilization compared to racemic ketoprofen exposure. The growth inhibition test showed that racemic ketoprofen and dexketoprofen exhibited different effect concentration values (240.2 and 65.6 µg/L, respectively). Furthermore, racemic ketoprofen and dexketoprofen did not exert cytotoxic effects in PLHC-1 cells and produced compound-, time-, and concentration-specific differential effects on cytochrome P450 1A (CYP1A) and glutathione S-transferase levels. For CYP1A, the effects of racemic ketoprofen and dexketoprofen differed at the transcriptional and catalytic levels. Exposure to racemic ketoprofen and dexketoprofen modulated MRP1 and MRP2 mRNA levels, and these effects were also dependent on compound, exposure time, and concentration of the individual drug. The present study revealed for the first time the interactions between these NSAIDs and key detoxification systems and different sensitivity to the racemic mixture compared to its enantiomer. Environ Toxicol Chem 2018;37:201-212. © 2017 SETAC.

The serpulid Ficopomatus enigmaticus () as candidate organisms for ecotoxicological assays in brackish and marine waters.

Ficopomatus enigmaticus is an ubiquitous fouling reef-forming species, easy to sample and recognize, diecious with gamete spawning along different seasons in different salinity conditions. Due to its characteristics it could become a good candidate for the monitoring of both marine and brackish waters. The suitability of F. enigmaticus as a promising model organism in ecotoxicological bioassays was evaluated by a sperm toxicity and a larval development assay. The fertilization rate in different salinity conditions (range 5-35‰) was first assessed in order to detect the salinity threshold within which profitably perform the assays. Afterward copper (Cu2+), cadmium (Cd2+), sodium dodecyl sulfate (SDS) and 4-n-nonylphenol (NP) were used as reference toxicants in exposure experiments with spermatozoids (sperm toxicity assay) and zygotes (larval development assay). A dose-response effect was obtained for all tested toxicants along all salinity conditions except for 5‰ salinity condition where a too low (<30%) fertilization rate was observed. NP showed the highest degree of toxicity both in sperm toxicity and larval development assay. In some cases the results, expressed as EC50 values at 35‰ salinity condition, were similar to those observed in the literature for marine organisms such as the sea urchin (Paracentrotus lividus) and the marine serpulid Hydroides elegans, while the exposure of F. enigmaticus spermatozoids' to Cd2+ and NP resulted in toxicity effects several orders of magnitude higher than observed in P. lividus. Spermatozoids resulted to be slightly more sensitive then zygotes to all different toxicants.

Ecotoxicity of pristine graphene to marine organisms.

The ecotoxicity of pristine graphene nanoparticles (GNC1, PGMF) in model marine organisms was investigated. PGMF resulted more toxic than GNC1 to the bioluminescent bacterium Vibrio fischeri and the unicellular alga Dunaliella tertiolecta on the basis of EC50 values (end-points: inhibition of bioluminescence and growth, respectively). No acute toxicity was demonstrated with respect to the crustacean Artemia salina although light microscope images showed the presence of PGMF and GNC1 aggregates into the gut; a 48-h exposure experiment revealed an altered pattern of oxidative stress biomarkers, resulting in a significant increase of catalase activities in both PGMF and GNC1 1mg/L treated A. salina and a significant increase of glutathione peroxidase activities in PGMF (0.1 and 1mg/L) treated A. salina. Increased levels of lipid peroxidation of membranes was also observed in PGMF 1mg/L exposed A. salina.

Acute toxicity and biodegradability of N-alkyl-N-methylmorpholinium and N-alkyl-DABCO based ionic liquids.

N-alkyl-N-methylmorpholinium and N-alkyl substituted 1,4-diazabicyclo[2.2.2]octane (DABCO) based ionic liquids (ILs), N-alkyl-DABCO, bearing short alkyl chains are characterised by a low toxicity to Vibrio fischeri, although toxicity significantly increases on increasing the alkyl chain length. Alkyl chain length affects also biodegradability in the 28 days tests; the higher level of biodegradation was found in both the series in the case of the ethyl (C2) derivatives. In the case of N-ethyl DABCO based IL, although biodegradability is still around 40%, and consequently this IL cannot be classified as "readily biodegradable", this value is similar to the more biodegradable functionalized imidazolium based ILs.

Acute toxicity of ionic liquids for three freshwater organisms: Pseudokirchneriella subcapitata, Daphnia magna and Danio rerio.

The static acute toxicities of 18 ionic liquids (ILs) were determined for three representative freshwater organisms, the cladoceran Daphnia magna, the green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), and the fish Danio rerio (formerly known as zebrafish). The test kit compounds contained three widely used ILs (1-butyl-3-methylimidazolium bis(triflimide), [bmim][Tf(2)N], butylpyridinium bis(triflimide), [bpy][Tf(2)N], and N,N-methylbutylpyrrolidinium bis(triflimide), [bmpyrr][Tf(2)N]) and 15 less common salts. These latter comprised a range of five anions, four positively charged head groups (ammonium, morpholinium, thiophenium, and sulfonium), five 1-methyl-3-alkyl imidazolium derivatives bearing a specific functional group on the longer alkyl chain (Cl, OH, or (CH(3))(3)Si) and three imidazolium derivatives characterized by the presence of a hydrogen atom on the imidazolium nitrogen ("Brønsted acidic imidazolium"-based ILs). Generally, long-chain ammonium salts showed higher toxicity to algae, cladocerans, and fish, whereas very low toxicities characterized sulfonium- and morpholinium-based ILs. In imidazolium-based ILs, the substitution of one or two carbon atoms of the longer alkyl chain with a more electronegative atom (chlorine or oxygen) reduced the acute toxicity for algae and cladocerans. Low toxicity also characterized the "Brønsted acidic imidazolium"-based ILs. Structural information for a rational designer of safer ILs can be obtained from these studies.

Tolerance of benzalkonium chloride, formalin, malachite green, and potassium permanganate in goldfish and zebrafish.

OBJECTIVE: To determine tolerance of goldfish and zebrafish to benzalkonium chloride, formalin, malachite green, and potassium permanganate. DESIGN: Tolerance study. ANIMALS: Adult goldfish (Carassius auratus) and zebrafish (Danio rerio). PROCEDURES: Groups of fish (n = 10/group) were exposed to each disinfectant at the therapeutic dosage; at 0.25, 0.5, 3, and 5 times the concentration used for the therapeutic dosage; and at the concentration used for the therapeutic dosage but for 3 or 5 times the recommended exposure time. RESULTS: In both species, exposure to malachite green at the therapeutic dosage resulted in toxic effects, including death. Exposure to formalin at the therapeutic dosage resulted in toxic effects in goldfish, but not zebrafish, and exposure to potassium permanganate resulted in toxic effects in zebrafish, but not goldfish. On the basis of the ratio of therapeutic dosage to median lethal dosage, in goldfish, formalin was more toxic than benzalkonium chloride, which was more toxic than malachite green, which was more toxic than potassium permanganate. In zebrafish, potassium permanganate was more toxic than formalin and benzalkonium chloride, which were approximately equally toxic and more toxic than malachite green. Extending treatment time increased the toxicity of potassium permanganate in zebrafish and the toxicity of formalin and malachite green in goldfish, but did not alter the toxicity of the other disinfectants. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that there was no consistency between zebrafish and goldfish in their tolerance to disinfectants, and that therapeutic dosages reported in the literature for these disinfectants were not always safe.